This invention relates to the enzymatic synthesis of fluorinated sugars (including fluorinated amino or amido sugars) which have a cyclic structure and a side chain and hence have a nucleus of more than 6 carbon atoms. An aspect of this invention relates to the synthesis of fluorinated sialic acids or fluorinated nonulosaminic acids, a family of amino sugars containing at least 8, more typically at least 9, carbon atoms.
Another aspect of this invention relates to the enzymatic rid synthesis of 3-fluoroneuraminic acid (5-acetamido-3, 5-didesoxy-3-fluoro-D-glycero-D-galacto-nonulopyranosonic acid) and other 3,5-didesoxy-3-fluorononulopyranosonic acid derivatives and their use.
The sialic acids are generally cyclic pentoses (furanoses) or cyclic hexoses (pyranoses) with a side chain (typically three carbon atoms long); accordingly, the sugar nucleus of these compounds has 8 or 9 carbon atoms, and this nucleus can be substituted with an amino group which can be in turn be substituted with acyl groups and the like. The most important embodiments of this class of compounds can be considered to be derivatives of amino sugars such as neuraminic acid, C9H17NO8. At least five sialic acids occur in nature; they are widely distributed throughout the animal kingdom (including some bacteria as well as more complex organisms such as mammals) and appear to be regular components of glycoproteins and glycolipids (where they typically occupy terminal positions). The most important of the sialic acids is generally considered to be the N-acetyl derivative of neuraminic acid (xe2x80x9cNeu5Acxe2x80x9d), i.e. 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid.
Preparations of sialic acids having 8- as well as 9-carbon nuclei are described in the scientific literature. For an example of a synthesis involving a furanose such as D-arabinose, see U. Kragl et al, J. Chem. Soc. Perkin. Trans. 1:119-124 (1994).
Because Neu5Ac and similar sugars occur so widely in higher animals and are so intimately involved in the physiology of mammals, they can be used as diagnostic and investigative tools, provided that they are suitably labeled with an isotope which can be measured or detected by various imaging techniques, radioactivity measurements, and other non-invasive procedures.
Fluorine is one of the most important labeling elements. The stable isotope, 19F, provides opportunities for in vivo investigation with 19F-NMR spectroscopy and has advantages over 1H-NMR spectroscopy used in MR-tomography. In the body, fluorine occurs naturally only in teeth and bones. Therefore, it is possible to observe the kinetics of the biodistribution of fluorine-labeled compounds.
Fluorine has several isotopes in addition to 19F, all of which are unstable, but only one of these has practical significance: the isotope, 18F, which is radioactive and has the longest half-life of the unstable isotopes (the other unstable isotopes have half-lives lasting less than 3 minutes). The 18F isotope has a half-life of 110 minutes and is very useful in biological studies and in medicine, but a half-life of less than 2 hours does impose some limits on its utility. Examples of the uses of 18F include non-invasive measurement of pharmacokinetic phenomena and the localization of tumors with 18F-labeled 2-fluorodesoxyglucose (e.g. by positron emission tomography).
The short half-life of the 18F isotope can impose severe requirements upon methods for synthesizing the 18F-labeled compound. The yield of labeled compound should be high, and, even more important, the synthesis must be very rapid.
In any method in which an F-labeled compound is used, it is generally necessary that its physiological properties (e.g. its properties as a substrate for an enzyme) be similar to the endogenous, non-fluorinated compound it is supposed to mimic. The fluorine atom has the advantage of being fairly small in its covalent radius and hence does not differ too markedly from hydrogen in terms of steric hindrance. A fluorine substituent does differ from other substituents in terms of charge density, due to its high electronegativity and electronic density. But generally speaking, the advantages of fluorine as a labeling substituent far outweigh its disadvantages.
A number of F-labeled derivatives of Neu5Ac are known. For example, the 3-fluorine derivative (xe2x80x9cNeu5Ac3Fxe2x80x9d), which has the systemic name 5-acetamido-3,5-didesoxy-3-fluoro-D-glycero-D-galacto-nonulopyranosonic acid, can be prepared by the aldol condensation of N-acetylmannosamine (xe2x80x9cManNAcxe2x80x9d) and fluoropyruvate or xcex2-fluoropyruvic acid (Fxe2x80x94CH2xe2x80x94COxe2x80x94COOH, systemic name 1-fluoro-2-oxopropanoic acid). The yield, however, is moderate (1.5%) and the purification is labor intensive. A recent paper discloses an electrophilic selective fluoridation which provided yields up to 80% and diastereomer selectivity of 75%. But the need for a faster and more stereospecific synthesis still exists.
The biosynthesis of Neu5Ac is enzyme-catalyzed and would appear to provide a model for a quick synthesis. In the biosynthesis, ManNAc is reacted with the pyruvate (pyruvic acid, CH3xe2x80x94COxe2x80x94COOH), and the enzyme catalyst is N-acetylneuraminic acid aldolase. N-acetylneuraminic acid aldolase EC 4.1.3.3 can be found in animal tissue and some bacteria. This enzyme has also been produced by biotechnology methods involving common microorganisms such as E. coli. The natural sialic acid Neu5Ac has been made successfully by enzymatic synthesis, but the preparation of Neu5Ac derivatives (particularly Neu5Ac3F) is more problematic.
The enzymatic synthesis of Neu5Ac3F has been investigated. For example, the amounts of substrates (the sugar component, ManNAc, and the pyruvate, xcex2-fluoropyruvate) have been varied considerably. The ManNAc would be expected to react with the xcex2-fluoropyruvate (xe2x80x9cF-pyrxe2x80x9d) to form Neu5Ac F-substituted at the 3-position (Neu5Ac3F), but under the conditions chosen in the If literature (50 mM of both substrates, 0.1 U*/ml and N-acetylneuraminic acid aldolase [xe2x80x9cNeu5Ac-aldolasexe2x80x9d] from E. coli in water, buffered at a pH of 7.7, maintained at 37xc2x0 C. and incubated for 24 hours), no conversion to Neu5Ac3F was observed. (The expression U*, a convenient enzyme activity unit employed in presenting data hereafter, was devised by the enzyme supplier, Toyobo, and is the enzyme activity with respect to the standard reaction of xe2x80x9cPyrxe2x80x9d (pyruvate)+ManNAc to obtain Neu5Ac. The expression U [without the asterisk], also used hereafter, is the enzyme activity with respect to the reaction of F-Pyr+ManNAc to Neu5Ac3F.)
Accordingly, further investigation of the enzymatic synthesis of these fluorinated sugars (particularly fluorinated amino sugars such as Neu5Ac3F) is needed.
It has now been discovered that the manipulation of enzyme synthesis conditions can provide a high-yield and preferably rapid preparation of fluorinated xe2x89xa78-carbon sugars from the appropriate substrates using an aldolase enzyme. First and foremost, the enzyme concentration must be increased drastically in comparison to the concentration reported in the literature. This drastic increase in enzyme concentration does not create any serious economic drawback. The enzyme obtained through biotechnology methods, e.g. from E. Coli, is relatively inexpensive. The enzyme Neu5Ac-aldolase from C. perfringens is also commercially available and can be used in this invention with results similar to the enzyme from E. coli. Moreover, the enzyme exhibits remarkable stability and can be recovered and reused in subsequent cycles or batches.
To insure that the synthesis will be fast enough to permit the use of 18F-labeled compounds, the sugar component is preferably present, in molar terms, in a large excess by comparison to the fluorinated component. The fluorinated component concentration should be sufficient also, however, and it is preferred, in the synthesis of 18F-labeled compounds, to introduce a significant amount of 19F-containing substrate as a carrier.
For cost-effectiveness, the synthesis is carried out on a scale in which the amounts of substrates are in the tens or hundreds of millimoles. Aldolase enzyme activity peaks within the range of 30-60 mM of F-pyr and generally increases with increasing amounts (e.g.  greater than 300, preferably  greater than 400 mM) of the sugar. The aldolase enzyme concentration is preferably in excess of 1 U*/ml and can range as high as 2,500 U*/ml. When the amount of F-Pyr is within the desired range (e.g. about 50 mM), the preferred aldolase enzyme concentration is 20 to 500 U*/ml, 100 to 200 U*/ml being especially preferred. Further, the reaction can also depend on temperature and pH. The preferred temperature range from about 1xc2x0 C. to about 55xc2x0 C. The preferred pH can range from about 5 to about 10.